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Method for constructing suicide plasmid and bacterial strain capable of expressing fluorescence based on is26 insertion sequence

An IS26 and insertion sequence technology, applied in the field of genetic engineering, can solve problems such as interference experiments, heavy workload, and cumbersome experimental operations

Active Publication Date: 2021-02-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Generally, this method can only construct engineering plasmids. When it is necessary to endow clinical wild large plasmids with a fluorescent gene, these methods will be subject to various limitations and the success rate is low.
In addition, engineering plasmids will exist and have been lost, and poor stability may interfere with subsequent experiments
In addition, there is a disadvantage in the above two constructions of fluorescence, that is, the experimental operation is cumbersome, the workload is heavy, the cost is high, the throughput is low, and a large number of fluorescent strains cannot be constructed at one time.

Method used

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  • Method for constructing suicide plasmid and bacterial strain capable of expressing fluorescence based on is26 insertion sequence
  • Method for constructing suicide plasmid and bacterial strain capable of expressing fluorescence based on is26 insertion sequence
  • Method for constructing suicide plasmid and bacterial strain capable of expressing fluorescence based on is26 insertion sequence

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Embodiment 1

[0086] (1) A method for constructing fluorescent strains based on IS26 insertion sequence transposition mutation technology, such as figure 1 shown. The present invention combines suicide type R6k replicon, lux gene cluster, IS26 insertion sequence, tellurite resistance gene (tpm R ), combined with the RP4 conjugative transfer gene to construct a new plasmid p26-RP4-lux. like figure 2 Shown, the construction process of the p26-RP4-lux plasmid that the present invention comprises mainly is:

[0087] The pUC26-tpm plasmid (purchased from Shanghai Jikai Gene Technology Co., Ltd.) and the pUC26-RP4 plasmid (purchased from Shanghai Jikai Gene Technology Co., Ltd.) were respectively digested with AflII enzyme and XhoI enzyme from NEB Company, and agarose gel electrophoresis enzyme The digested products were purified by TAKARA Gel Recovery Kit (recovered fragments were 2880bp and 4702bp), and the purified digested products were catalyzed by TAKARA T4 DNA ligase and ligated overni...

Embodiment 2

[0109] The construction method of the p26-RP4-lux plasmid is the same as in Example 1. In this embodiment, 20 clinical bacterial strains are randomly selected as bacterial strains to be transformed. These 20 bacterial strains are constructed by our method for constructing fluorescent bacterial strains (same as in Example 1). E.coli WM3064(p26-RP4-lux) and target strains (Table 1) were subjected to conjugation transfer experiments, the results are as follows Figure 6 and as shown in Table 1, Figure 6 It is a picture of the successfully transformed fluorescent strains. 18 fluorescent strains have been successfully constructed, and the transformation success rate is 90%. Then, the 18 fluorescent strains that were successfully constructed were subjected to a second conjugation transfer experiment with the recipient strain E.coli C600, and a fluorescent zygote could be obtained, indicating that the IS26 transposable unit was inserted into the plasmid. If the fluorescent zygote co...

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Abstract

The invention discloses a method for constructing a suicide plasmid capable of expressing fluorescence and a bacterial strain based on an IS26 insertion sequence. The construction method of the suicide plasmid is as follows: pUC26-tpm plasmid and pUC26-RP4 plasmid are digested by AflII and XhoI, and then the recovered product after electrophoresis is connected with ligase to construct p26-RP4 plasmid; Digest p26-RP4 plasmid and pBBR1MCS4-LuxCDAB plasmid, and then connect the product recovered after electrophoresis with ligase to construct a suicide plasmid capable of expressing fluorescence. The suicide plasmid obtained in the present invention is transformed into Escherichia coli WM3064 competent cells to obtain fluorescently labeled Escherichia coli; finally, it is co-cultured with the target strain to construct a fluorescent strain. The method of the invention has high efficiency and can be used for constructing drug-resistant strains, evaluating pharmacokinetics and pharmacodynamics or screening drugs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for constructing a suicide plasmid capable of expressing fluorescence and a bacterial strain based on an IS26 insertion sequence. Background technique [0002] To construct bacteria that can emit light, with the help of fluorescence imaging system or animal imaging system, the movement of bacteria and gene behavior in living organisms can be directly observed, and the progress of disease development can be observed and the evaluation of pharmacokinetics and pharmacodynamics can be performed. Fluorescently labeled bacteria and the technology of constructing luminescent bacteria can provide us with a new platform for studying the dynamic changes of bacteria under environmental stimuli, gene exchange, and new drug development. For example: (Yang Fan et al., 2014) transferred a plasmid containing the Lux gene cluster into a strain of Escherichia coli, and then st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/74C12N15/66C12N15/65C12N1/21C12R1/19C12R1/22C12R1/42
CPCC12N15/65C12N15/66C12N15/70C12N15/74C12N2800/90
Inventor 孙坚何玉张李龚刘苹江颖琳许金霞刘雅红廖晓萍
Owner SOUTH CHINA AGRI UNIV